Homologously recombinant slow growing mycobacteria and uses therefor

ABSTRACT

A method of transforming slow-growing mycobacteria, such as M. bovis BCG, M. leprae, M. tuberculosis M. avium, M. intracellulare and M. africanum; a method of manipulating genomic DNA of slow-growing mycobacteria through homologous recombination; a method of producing homologously recombinant (HR) slow-growing mycobacteria in which heterologous DNA is integrated into the genomic DNA at a homologous locus; homologously recombinant (HR) slow-growing mycobacteria having heterologous DNA integrated into their genomic DNA at a homologous locus; and mycobacterial DNA useful as a genetic marker.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/711,334,filed Jun. 6, 1991, entitled "Recombinant BCG-HIV Vaccines", nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/367,894,filed Jun. 19, 1989, entitled "Vector-Mediated Genomic Insertion andExpression of DNA in BCG", now abandoned, and the correspondingInternational Application PCT/US90/03451, filed Jun. 18, 1990, entitled"Vector-Mediated Genomic Insertion and Expression of DNA in BCG"; andthe International Application PCT/US89/02962, filed Jul. 7, 1989,entitled "Recombinant Mycobacterial Expression Vehicles and UsesTherefor," which are/were combined and claimed priority to three U.S.applications, U.S. Ser. No. 07/361,944, filed Jun. 5, 1989, entitled"Recombinant Mycobacterial Vaccine", now U.S. Pat. No. 5,504,005, whichis a continuation-in-part of U.S. Ser. No. 07/223,089, filed Jul. 22,1988, entitled "Stable Expression of Cloned Genes in Mycobacteria UsingPhage and Plasmid Vectors", now abandoned, and of U.S. Ser. No.07/216,390, filed Jul. 7, 1988, entitled "Recombinant MycobacteriaHaving DNA of Interest Stably Integrated Into Genomic DNA", nowabandoned, which are continuation-in-part applications of U.S. Ser. No.07/163,546, filed Mar. 3, 1988, entitled "Recombinant MycobacterialVaccine", now abandoned, and the corresponding International ApplicationPCT/US88/00614, filed Feb. 29, 1988, entitled "Recombinant MycobacterialVaccine"; which is a continuation-in-part of U.S. Ser. No. 07/020,451,filed Mar. 2, 1987, entitled "Recombinant Mycobacterial Vaccine", nowabandoned. The teachings of these related applications are incorporatedherein by reference. This application is also related to U.S. Ser. No.08/096,027, filed Jul. 22, 1993, now U.S. Pat. No. 5,591,632, entitled"Recombinant BCG Vaccines."

BACKGROUND OF THE INVENTION

The World Health Organization estimates that one in three human beingsis believed to be infected with Mycobacterium tuberculosis (Styblo, K.,Reviews of Infectious Diseases. Vol. II, Suppl. 2, March-April, 1989;Bloom and Murray, Science 257:1055-1067, 1992). Over the past decade,there has been a recent resurgence in the incidence of tuberculosis indeveloped countries that has coincided with the AIDS epidemic (Sniderand Roper, N. England J. Med. 326:703-705 (1992)). Because of theirimpact as major human pathogens and as a result of their profoundimmunostimulatry properties, mycobacteria have long been intensivelystudied. In the early 1900s, an attenuated mycobacterium,Mycobacerium(M.) bovis Bacille Calmette-Guerin (M. bovis BCG or BCG),was isolated for use as a vaccine against tuberculosis (Calmette et al.Acad. Natl. Med. (Paris), 91:787-796, 1924; reviewed in Collins, F. M.,Bacterial Vaccines (R. Germanier, ed.), Academic Press, pp. 373-418,1984). Although the efficacy of this vaccine against tuberculosis variedconsiderably in different trials, and the reasons for its variableefficacy have yet to be resolved, BCG is among the most widely usedhuman vaccines (Luelmo, F., Am. Rev. Respir. Dis. 125:70-72, 1982; Fine,P. E. M., Reviews of Infectious Diseases II (supp. 2), 5353-5359, 1989).

The recent application of molecular biological technology to the studyof mycobacteria has led to the identification of many of the majorantigens that are targets of the immune response to infection bymycobacteria (Kaufmann, S. H. E., Immunol. Today 11:129-136, 1990;Young, R. A., Ann. Rev. Immunol. 8:401-420, 1990; Young et al., AcademicPress Ltd., London, pp. 1-35, 1990; Young et al., Mol. Microbiol.6:133-145, 1992)) and to an improved understanding of the molecularmechanisms involved in resistance to antimycobacterial antibiotics(Zhang et al., Nature 358:591-593, 1992; Telenti et al., Lancet341:647-650, 1993). The development of tools that permit moleculargenetic manipulation of mycobacteria has also allowed the constructionof recombinant BCG vaccine vehicles (Snapper et al., Proc. Natl. Acad.Sci. USA 85:6987-6991, 1988; Husson et al., J. Bacteriol. 172:519-524,1990; Martin et al., B. Nature 345:739-743, 1990; Snapper et al., Mol.Microbiol. 4:1911-1919, 1990; Aldovini and Young, Nature 351:479-482,1991; Jacobs et al., Methods Enzymol. 204:537-555, 1991; Lee et al.,Proc. Natl. Acad. Sci. USA 88:3111-3115, 1991; Stover et al., Nature351: 456-460, 1991; Winter et al., Gene 109:47-54, 1991; Donnelly-Wu etal., Mol. Microbiol. 7:407-417, 1993)). Genome mapping and sequencingprojects are providing valuable information about the M. tuberculosisand M. leprae genomes that will facilitate further study of the biologyof these pathogens (Eiglmeier et al., Mol. Microbiol., in press, 1993;Young and Cole, J. Bacteriol. 175:1-6, 1993).

Despite these advances, there are two serious limitations to our abilityto manipulate these organisms genetically. First, very few mycobacterialgenes that can be used as genetic markers have been isolated(Donnelly-Wu et al., Mol. Microbiol. 7:407-417, 1993)). In addition,investigators have failed to obtain homologous recombination in slowgrowing mycobacteria, such as M. tuberculosis and M. bovis BCG (Kalpanaet al., Proc. Natl. Acad. Sci. USA 88:5433-5447, 1991; Young and Cole,J. Bacteriol. 175:1-6, 1993)), although homologous recombination hasbeen accomplished in the fast growing Mycobacterium smegmatis (Husson etal., J. Bacteriol. 172: 519-524, 1990)).

SUMMARY OF THE INVENTION

Described herein is a method of transforming slow-growing mycobacteria,such as M. bovis BCG, M. legrae, M. tuberculosis M. avium, M.intracellulare and M. africanum; a method of manipulating genomic DNA ofslow-growing mycobacteria through homologous recombination; a method ofproducing homologously recombinant (HR) slow-growing mycobacteria inwhich heterologous DNA is integrated into the genomic DNA at ahomologous locus; homologously recombinant (HR) slow-growingmycobacteria having heterologous DNA integrated into their genomic DNAat a homologous locus; and mycobacterial DNA useful as a genetic marker.

Applicants have succeeded in introducing heterologous DNA into (i.e.,transforming) slow-growing mycobacteria through the use ofelectroporation in water (rather than in buffer). In the present methodof transforming slow-growing mycobacteria, heterologous DNA (such aslinear DNA or plasmid DNA) and slow-growing mycobacteria (e.g., M. bovisBCG, M. leprae, M. tuberculosis M. avium, M. intracellulare and M.africanum) are combined and the resulting combination is subjected toelectroporation at an appropriate potential and capacitance forsufficient time for the heterologous DNA to enter the slow growingmycobacteria, resulting in the production of transformed mycobacteriacontaining the heterologous DNA. In one embodiment, heterologous DNA andM. bovis BCG are combined and subjected to electroporation in water. Ina particular embodiment, the M. bovis BCG-heterologous DNA combinationis subjected to electroporation in water at settings of approximately2.5 kV potential and approximately 25 μF capacitance. Optionally, priorto harvest, cells to be transformed are exposed to glycine (such as byadding 1-2% glycine to culture medium in which the slow-growmycobacteria are growing) in order to enhance or improve transformationefficiencies. In one embodiment, 1.5% glycine is added to the culturemedium 24 hours prior to harvesting of the cells, which are thencombined with heterologous DNA to be introduced into the slow-growingmycobacteria. The resulting combination is subjected to electroporation,preferably in water, as described above.

In a further embodiment of the method of transforming slow growingmycobacteria, cultures of the cells are maintained in (continuouslypropagated in) mid-log growth, in order to increase the fraction ofcells which are undergoing DNA synthesis (and which, thus, are competentto take up heterologous DNA). Cultures of cells maintained in log-phasegrowth are subjected to electroporation, preferably in water and, as aresult, are transformed with the heterologous DNA. As described above,efficiency of transformation can be increased by exposing theslow-growing mycobacteria to glycine prior to electroporation. Thus, inthis embodiment, slow-growing mycobacteria in log-phase growth arecombined with heterologous DNA (e.g., plasmid DNA, linearized DNA) to beintroduced into the slow-growing mycobacteria. The resulting combinationis subjected to electroporation (preferably in water), under conditions(potential and capacitance settings and sufficient time) appropriate fortransformation of the cells. Optionally, prior to electroporation, thelog-phase cells are exposed to glycine (e.g., approximately 1-2% glycineadded to culture medium) in order to enhance transformation efficiency.

Heterologous DNA introduced into slow-growing mycobacteria is DNA fromany source other than the recipient mycobacterium. It can be homologousto DNA present in the recipient mycobacterial genomic DNA, nonhomologousor both. DNA which is homologous to mycobacterial genomic DNA isintroduced into the genomic DNA by homologous recombination orintegration. Alternatively, the heterologous DNA introduced by thepresent method can be nonhomologous and, thus, enter mycobacterialgenomic DNA by random integration events or remain extrachromosomal(unintegrated) after it enters the mycobacterium. In addition, in oneembodiment of the present method, non-homologous DNA linked to orinserted within DNA homologous to genomic DNA of the recipientmycobacterium is introduced into genomic DNA of the recipientmycobacterium as a result of homologous recombination which occursbetween genomic DNA and the homologous DNA to which the nonhomologousDNA is linked (or in which it is inserted). For example, as describedherein, a mycobacterial gene which encodes a genetic marker has beenidentified and isolated and used to target homologous integration ofheterologous DNA (DNA homologous to genomic DNA of the mycobacterialrecipient, alone or in conjunction with DNA not homologous to genomicDNA of recipient mycobacteria) into genomic DNA of a slow-growingmycobacterium. Specifically, the M. bovis BCG gene encoding orotidine-5-monophosphate decarboxylase (OMP DCase) (uraA) has been isolated, as hasDNA flanking OMP DCase. The OMP DCase gene and the flanking DNA havebeen sequenced. The mycobacterial DNA containing the uraA locus,modified to contain heterologous DNA (a selectable marker gene) has beenused to carry out integration of the heterologous DNA (the mycobacterialDNA and the selectable marker gene) into mycobacterial genomic DNA,resulting in production of homologously recombinant mycobacteriacontaining the heterologous DNA of a homologous locus. Specifically, M.bovis BCG DNA containing the uraA locus and flanking sequences wasmodified to replace the OMP DCase coding sequence with the Kan^(r)selectable marker gene (aph). The resulting construct, which includedapproximately 1.5 kb uraA flanking sequences on each side of theselectable marker gene, was transformed into M. bovis BCG, using themethod described above. M. bovis BCG cultures in mid-log growth weresubjected to electroporation in water, resulting in transformation ofcells with the construct. Transformants were selected for further study,which showed that all transformants assessed contained vector DNAintegrated into the genome and that in some of the transformants, thetransforming DNA had integrated at the homologous genomic locus. Thus,heterologous DNA of interest has been introduced into genomic DNA ofslow-growing mycobacteria through homologous recombination, to producehomologously recombinant slow-growing mycobacteria in which theheterologous DNA is integrated into the homologous genomic locus (agenomic locus homologous to at least a portion of the heterologous DNA).

Heterologous DNA which includes DNA homologous to genomic DNA of therecipient mycobacterium (homologous DNA) and DNA which is not homologousto genomic DNA of the recipient mycobacterium (nonhomologous DNA) can beintroduced into (transformed into) slow growing mycobacterium by thepresent method for several purposes. As described herein, heterologousnonhomologous DNA encoding a product to be expressed by the resultinghomologously recombinant slow-growing mycobacterium has been introducedinto mycobacterial genomic DNA at a locus homologous with additionalsequences to which the nonhomologous DNA is linked. In this embodiment,the DNA construct transformed into recipient slow-growing mycobacteriacomprises homologous DNA, which directs or targets introduction of theheterologous DNA into the homologous locus of the mycobacterial genome,and nonhomologous DNA, which is expressed in transformed homologouslyrecombinant mycobacteria. In this embodiment, the nonhomologous DNA isintroduced into mycobacterial genomic DNA in such a manner that it isadded to the genomic DNA or replaces genomic DNA. In a secondembodiment, heterologous DNA integrated into genomic DNA is notexpressed in the recipient cells. In this embodiment, the DNA constructincludes homologous DNA for targeting into a homologous genomic locusand DNA which acts to knock out (inactivate) or activate a residentmycobacterial gene. In the case of inactivation, the mycobacterial geneis "knocked out", in the sense that it is rendered inactive by additionof DNA whose presence interferes with its ability to function, byremoval or replacement of sequences necessary for it to be functional orby its complete removal from the mycobacterial genome. In the case ofactivation, the heterologous DNA integrated into the genomic DNA turnson or enhances expression of a mycobacterial gene, such as byintroducing a heterologous promoter which controls the mycobacterialgene expression. In the embodiment in which heterologous DNA affectsexpression of an endogenous mycobacterial gene, the homologous DNA canserve both functions (i.e., the targeting and inactivation/activatingfunctions); if that is the case, the DNA construct includes onlyhomologous DNA. Alternatively, the DNA construct can include homologousDNA (for targeting purposes) and nonhomologous DNA (for alteringfunction of the mycobacterial gene).

Homologously recombinant slow-growing mycobacteria of the presentinvention are useful, for example, as vehicles in which proteins encodedby the heterologous nonhomologous DNA are expressed. They are useful asvaccines, which express a polypeptide or a protein of interest (or morethan one polypeptide or protein), such as an antigen or antigens of oneor more pathogens against which protection is desired (e.g., to preventor treat a disease or condition caused by the pathogen). Pathogens ofinterest include viruses, retroviruses, bacteria, mycobacteria, othermicroorganisms, organisms or substances (e.g., toxins or toxoids) whichcause a disease or condition to be prevented, treated or reversed. Thehomologously recombinant slow-growing bacteria can also be used toexpress enzymes, immunopotentiators, lymphokines, pharmacologic agents,antitumor agents (e.g., cytokines), or stress proteins (useful forevoking or enhancing an immune response or inducing tolerance in anautoimmune disease). For example, homologously recombinant slow-growingmycobacteria of the present invention can express polypeptides orproteins which are growth inhibitors or are cytocidal for tumor cells(e.g., interferon α, β or γ, interleukins 1-7, tumor necrosis factor(TNF) α or β) and, thus, are useful for treating certain human cancers(e.g., bladder cancers, melanomas). Homologously recombinantslow-growing mycobacteria of the present invention are also usefulvehicles to elicit protective immunity in a host, such as a human orother vertebrate. They can be used to produce humoral antibody immunity,cellular immunity and/or mucosal or secretory immunity. The antigensexpressed by the homologously recombinant slow-growing mycobacteria,useful as vaccines or as diagnostic reagents, are also the subject ofthe present invention. In addition, homologously recombinantslow-growing mycobacteria of the present invention are useful asvaccines in which the heterologous DNA introduced through homologousintegration is not itself expressed, but acts to knock out amycobacterial gene necessary for pathogenicity of the slow-growingmycobacterium or its growth in vivo. Such homologously recombinantslow-growing mycobacteria are useful as vaccines to provide protectionagainst diseases caused by the corresponding wild-type mycobacterium oras a vaccine vehicle which contains a gene(s) encoding an antigen(s) ofa different pathogen(s) (e.g., as a vaccine to provide protectionagainst an organism other than the corresponding wild-type mycobacteriumor against a toxin or toxoid).

The vaccine of the present invention has important advantages overpresently available vaccines. For example, mycobacteria have adjuvantproperties; they stimulate a recipient's immune system to respond toother antigens with great effectiveness. In addition, the mycobacteriumstimulates long-term memory or immunity. This means that a single (onetime) inoculation can be used to produce long-term sensitization toprotein antigens. Long-lasting T cell memory, which stimulates secondaryantibody response neutralizing to the infectious agent or toxic. This isparticularly useful, for example, against tetanus and diphtheria toxins,pertussis, malaria, influenza, herpes viruses and snake venoms.

BCG in particular has important advantages as a vaccine vehicle. Forexample, it can be used repeatedly in an individual and has had a verylow incidence of adverse effects. In addition, BCG, as well as othermycobacteria, have a large genome (approximately 3×10⁶ bp in length). Asa result, a large amount of heterologous DNA can be accommodated within(incorporated into) the mycobacterial genome, which means that a largegene or multiple genes (e.g., DNA encoding antigens for more than onepathogen) can be inserted into genomic DNA, such as by homologousrecombination.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a structural and functional map of the M. bovis BCG uraAlocus, in which a restriction map of the uraA locus and the recombinantinsert DNAs for several plasmids used to study this region are depicted.The relative positions of the BCG uraA gene and the portions of othergenes identified are summarized graphically and the ability of eachrecombinant to complement the E. coli pyrF mutant is indicated.

FIGS. 2A-2D is the nucleic acid sequence of the BCG uraA locus (Seq IDNo. 1) and the predicted protein products (Seq ID No. 2).

FIG. 3 is a schematic representation of integration by homologousrecombination in BCG. The uraA locus in wild-type BCG (top), thetransforming DNA (middle) and a BCG transformant in which thetransforming DNA fragment has integrated via homologous recombination(bottom) are represented.

FIG. 4 is a schematic representation of the Southern analysis of the BCGtransformant represented in FIG. 3.

FIG. 5 shows the results of Southern blot analysis of genomic DNAisolated from wild-type BCG (WT) and a BCG transformant (6015-9). Thepositions of DNA markers are indicated to the right and the apparentsize of each of the hybridizing DNA bands is indicated to the left.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, Applicants have demonstrated introduction ofheterologous DNA into slow-growing mycobacteria (transformation ofheterologous DNA into slow-growing mycobacteria) and incorporation ofheterologous DNA at a homologous locus in genomic DNA of slow-growingmycobacterial (integration of heterologous DNA into the genomic DNAthrough homologous recombination). As a result, they have producedhomologously recombinant slow-growing mycobacteria having heterologousDNA integrated at a homologous locus in their genomic DNA. Inparticular, as described herein, Applicants have introduced heterologousDNA into M. bovis BCG (BCG) and demonstrated that it is present in theresulting homologously recombinant BCG at a genomic location homologouswith sequences present in the DNA construct transformed into the BCG.The DNA construct introduced into BCG by the method described hereinincluded heterologous DNA containing the uraA locus (homologous DNA) andnonhomologous DNA (a selectable marker gene); the heterologoushomologous DNA flanked the nonhomologous DNA in the construct. Both theheterologous homologous DNA and the heterologous nonhomologous DNA inthe DNA construct were shown to have integrated into the genome of therecipient mycobacterial cells at a homologous locus (a genomic locushomologous with the DNA including and/or flanked by the homologous DNAin the DNA construct).

As a result of the work described herein, a method of transforming slowgrowing mycobacteria, a method of introducing heterologous DNA intogenomic DNA of a slow growing mycobacterium through integration at ahomologous locus, DNA constructs useful in the method of introducingheterologous DNA into a homologous locus in genomic DNA of a slowgrowing mycobacteria, homologously recombinant slow growing mycobacteriawhich contain heterologous DNA at a homologous locus in their genomicDNA, a BCG gene encoding orotidine-5'-monophosphate decarboxylase (BCGOMP DCase) and homologously recombinant slow growing mycobacteria usefulas vaccines are available. The following is a description of the presentmethod, DNA constructs and vaccines, as well as the isolated BCG OMPDCase gene and its use.

The present invention includes an improved method of transforming slowgrowing mycobacteria. In the present method, slow growing mycobacteriaare subjected to electroporation in water, preferably after exposure to(culturing in the presence of) glycine prior to electroporation andpreferably also while they are in mid-log growth. Slow growingmycobacteria to be transformed with heterologous DNA are combined withthe heterologous DNA (which can be plasmid/circular DNA or linear DNA)in water. The resulting combination is subjected to electroporationunder conditions (e.g., potential, capacitance and time) sufficient forentry of the heterologous DNA into the slow growing mycobacteria.Electroporation is carried out at approximately 2 to 2.5 kV potentialand approximately 1 to 125 μF capacitance for approximately 4 to 40milliseconds. In a specific embodiment, slow growing mycobacterial cellsare electroporated in water at approximately 2.5 kV potential andapproximately 25 μF capacitance for 5-6 milliseconds. In a furtherembodiment, slow growing mycobacteria to be transformed are exposed toglycine (e.g., 1 to 2% glycine) by addition of glycine to culture mediumprior to harvest of the cells. In a particular embodiment, slow growingmycobacteria are exposed to 1.5% glycine, which is added to culturemedium, for approximately 24 hours prior to harvest of the cells fortransformation. In another embodiment, slow-growing mycobacteria are inmid-log growth when they are transformed. The cells can also have beenexposed to glycine, as described above, prior to electroporation,although that is not necessary. The mid-log slow growing mycobacteriaare combined with heterologous DNA to be introduced into them andsubjected to electroporation in water, as described above, resulting intransformation of the heterologous DNA into slow growing mycobacteria inthe combination.

The heterologous DNA introduced into slow growing mycobacteria by thepresent method is DNA obtained from any source other than themycobacterium into which it is being introduced. It can be of viral,bacterial, mycobacterial, invertebrate or vertebrate (including humanand other mammalian) origin, can be obtained from other organisms, suchas parasites, or can be produced to have the same nucleic acid sequenceas the DNA in its naturally occurring source. Alternatively, it can bemodified DNA. The DNA introduced can be plasmid (circular) DNA or linearDNA. The heterologous DNA contains DNA homologous to a locus in genomicDNA of the recipient slow growing mycobacteria, DNA nonhomologous to alocus in genomic DNA of the recipient cells or both. It is possible tocombine slow growing mycobacteria and a DNA construct in which theheterologous DNA is only nonhomologous DNA and carry out the presentmethod of transformation, if the goal is to transform slow growingmycobacteria with greater efficiency than is possible with existingmethods. Heterologous DNA introduced in this manner will integraterandomly into genomic DNA.

In order to produce homologously recombinant slow growing mycobacteriathrough homologous integration between mycobacterial genomic DNA andheterologous DNA, the DNA construct must include sufficient DNAhomologous with mycobacterial DNA to cause integration of the constructinto a homologous genomic locus. If only homologous DNA is present inthe DNA construct used (e.g., in a construct introduced in order toknock out or activate endogenous mycobacterial DNA), at least 400 bp ofhomologous DNA will generally be used. If the DNA construct includeshomologous DNA (for directing or targeting introduction intomycobacterial genomic DNA) and nonhomologous DNA (e.g., DNA encoding aproduct to be expressed in homologously recombinant slow growingmycobacteria), there is homologous DNA on both sides of (flanking bothends of) the nonhomologous DNA. In general, there will be at leastapproximately 250 bp of homologous DNA on each side of the nonhomologousDNA, although shorter flanking homologous sequences can be used,provided that they are of sufficient length to undergo homologousrecombination with genomic sequences, resulting in their introductioninto mycobacterial genomic DNA (alone or in conjunction withnonhomologous DNA with which the homologous DNA is present in the DNAconstruct). In the embodiment described in the examples, 1.5 kb ofhomologous DNA (1.5 kb of uraA flanking sequence) has been shown toresult in homologous integration, along with nonhomologous DNA, into theuraA locus of M. bovis BCG.

The homologous DNA present in the DNA construct can be any DNAhomologous to DNA present in genomic DNA of the recipient slow growingmycobacterium. Specifically described herein is the isolation andsequencing of the M. bovis BCG OMP DCase gene (uraA) and its use tointroduce heterologous nonhomologous DNA into M. bovis BCG genomic DNAat a homologous locus. As described in the examples, a BCG DNA fragmentwhich included the OMP DCase coding sequence was modified to remove theOMP DCase coding sequence and replace it with heterologous nonhomologousDNA encoding a selectable marker gene (i.e., the Kan^(r) (aph) gene).Specifically, the DNA construct was made by removing the OMP DCasecoding sequence from a 4.4 kb BCG DNA fragment containing uraA andreplacing it with the Kan^(r) gene (aph), to produce a DNA fragment inwhich the selectable marker gene is flanked by 1.5 kb uraA DNA (todirect homologous recombination or integration of the homologous DNAand, along with it, the nonhomologous DNA into mycobacterial genomicDNA). All or a portion of the OMP DCase gene can be used, with similarmodifications, as a component of a DNA construct including otherheterologous nonhomologous DNA to be introduced into M. bovis BCGgenomic DNA at the uraA locus. Alternatively, other M. bovis BCG genescan be used as the heterologous homologous component of a DNA constructuseful for introducing heterologous nonhomologous DNA into themycobacterium. Similarly, DNA from other slow growing mycobacteria(e.g., M. leprae, M. tuberculosis, M. avium, M. africanum) can beincorporated into a DNA construct to be used for homologousrecombination in the respective slow growing mycobacteria.

The heterologous nonhomologous DNA in the DNA construct introduced intoslow growing mycobacteria by the present method can be any DNA which isexpressed in the slow-growing mycobacteria or which is not expressed inthe recipient mycobacteria but alters mycobacterial protein expressionor function. For example, the heterologous nonhomologous DNA can be DNAencoding an antigen(s) of a pathogen or pathogens. A pathogen is anyvirus, micro-organism, other organism or substance (e.g., toxins,toxoids) which causes a disease or undesirable condition. Homologouslyrecombinant slow growing mycobacteria which express a protein antigen(s)from malaria sporozoites, malaria merozoites, diphtheria toxoid, tetanustoxoid, Leishmania, Salmonella, M. africanum, M. intracellulare, M.avium, treponema, pertussis, herpes virus, measles virus, mumps,Shigella, Neisseria, Borrelia, rabies, poliovirus, humanimmunodeficiency virus (HIV), Simian immunodeficiency virus (SIV), snakevenom, insect venom or vibrio cholera can be produced using the methodof the present invention. Homologously recombinant M. bovis BCG, which,in a nonhomologously recombinant form, has long been successfullyadministered as a vaccine in humans can be used. The DNA encoding theprotein antigen(s) can be obtained from sources in which it naturallyoccurs or can be produced through known recombinant techniques or knownchemical synthetic methods. For example, the DNA can be produced bygenetic engineering methods, such as cloning or by the polymerase chainreaction (PCR).

A multipurpose or multifunctional vaccine (one which contains andexpresses heterologous DNA encoding antigens from more than onepathogen) can be produced by the present method. In this embodiment, oneor more DNA constructs are used to introduce heterologous homologous DNAand heterologous nonhomologous DNA (DNA encoding an antigen againstwhich protection is desired) into the slow growing mycobacterium. If oneconstruct is used, it includes DNA encoding the antigens of interest,flanked by homologous DNA sufficient for introduction of theheterologous DNA into a homologous locus in the mycobacterium. More thanone construct can be used; in this case, each includes homologous DNAand nonhomologous DNA encoding an antigen of interest. A multifunctionalvaccine of the present invention can be homologously recombinant BCGwhich contains, within its genomic DNA, a gene encoding an antigen forM. leprae, a gene encoding an antigen for M. tuberculosis, a geneencoding an antigen for malaria and a gene encoding an antigen forLeishmania; these sequences are flanked by heterologous sequenceshomologous with BCG DNA and are introduced into the BCG genome byhomologous integration.

It is not necessary that heterologous nonhomologous DNA be expressed byhomologously recombinant slow growing mycobacteria of the presentinvention or even that there be heterologous nonhomologous DNA present.For example, in one embodiment, heterologous nonhomologous DNA isincorporated into genomic DNA of slow growing mycobacteria for thepurpose of inactivating an endogenous mycobacterial gene, such as a genenecessary for the pathogenicity of the mycobacterium. Any gene involvedin metabolism necessary for pathogenicity of the slow growingmycobacterium (or for its growth in humans or other animals) but whoseabsence (e.g., from being knocked out) does not prevent it from beingcultured can be targeted for inactivation. For example, the AROA gene ofM. tuberculosis can be inactivated. In another embodiment, heterologousnonhomologous DNA is introduced in order to activate or turn on anendogenous mycobacterial gene. In either case, the heterologousnonhomologous DNA need not be expressed.

Heterologous DNA can be homologous DNA only; it is not necessary thatheterologous nonhomologous DNA be present. For example, homologous DNAcan be introduced into an endogenous mycobacterial gene (such as oneessential for the pathogenicity of a slow growing mycobacterium) inorder to disrupt or inactivate that gene. This is particularly useful inthose embodiments in which an attenuated or disabled mycobacterium isdesired, such as for use as a vaccine to elicit an immune responseagainst the mycobacterium itself or as a vehicle to be used in a similarmanner to that in which homologously recombinant BCG can be used (toexpress antigens of other pathogens).

Homologously recombinant slow growing mycobacteria of the presentinvention can be administered by known methods and a variety of routes(e.g., intradermally, intramuscularly, intravenously). They are usefulas vehicles in which the heterologous nonhomologous DNA is expressed andas modified slow grow mycobacteria (e.g., mycobacteria with reduced orabolished pathogenicity) which are disabled or attenuated and, thus,useful as vaccines.

The present invention will now be illustrated by the following examples,which are not to be considered limited in any way.

MATERIALS AND METHODS

Strains and plasmids. M. bovis BCG used for DNA isolation and subsequentconstruction of the recombinant BCG plasmid and λgt11 libraries was theMontreal Strain, ATCC #35735. M. bovis BCG was grown in Middlebrook 7H9media, supplemented with 0.05% Tween 80, as described in Aldovini andYoung, Nature 351:479-482, (1991). E. coli strain Y1107 (pyrF::Mu trpamlacZam hsdR- m+ su-) was obtained from D. Botstein. Plasmids werepropagated in the E. coli strain DH5α from Bethesda ResearchLaboratories. E. coli cultures used for plasmid selection were grown inLuria Bertani broth or agar with 50 μg/ml ampicillin. Phage M13 used forthe production of single stranded DNA were propagated in E. coli strainJM101 from New England BioLabs. JM101 was grown in YT medium (Maniatis).Genomic libraries were generated using pUC19 from Bethesda ResearchLaboratories. Plasmid pY6002 (Husson et al., J. Bacteriol., 172:519-5241990) was the source of the 1.3 kb BamHI DNA fragment containing theamino-glycoside phosphotransferase gene aph.

Enzymes. Klenow fragment of E. coli DNA polymerase was supplied byPromega. T7 polymerase, and Taq polymerase (Sequenase and Taquence) wereprovided by United States Biochemical.

Recombinant DNA library construction. To isolate BCG DNA, cells wereharvested by centrifugation, washed, and resuspended in 50 mM Tris (pH8.0), 10 mM EDTA, 10% sucrose, and 0.5 mg/ml lysozyme, and incubated at37 degrees for one hour. EDTA was then added to 1%, and the mixture wasincubated at room temperature for 15 minutes. Three phenol/chloroformextractions were performed, followed by RNase treatment,phenol/chloroform extraction, chloroform extraction and ethanolprecipitation. The DNA was then resuspended in TE buffer, (10 mM Tris pH7.5, imM EDTA).

To construct the plasmid library, the DNA was subjected to partialdigestion with Sau3A and DNA fragments of 2-6 kb were isolated byagarose gel electrophoresis onto DE81 paper and eluted in buffercontaining 10 mM Tris, HC1, 1M NaCl and 1 mM EDTA. The DNA fragmentswere then phenol-chloroform extracted, ethanol precipitated and ligatedinto BamH1 digested, calf-intestinal phosphatase treated pUC19 plasmidvector. E. coli cells were transformed with the ligated mixture, andapproximately 4×10⁵ recombinants were obtained. Plasmid DNA was obtainedfrom the pool of transformed colonies using an alkaline lysis method.

The λgt11 library was constructed using a procedure described by Young.(Young, R. A., et al., Proc. Natl. Acad. Sci., USA, 82:2583-2587(1985)). Briefly, BCG genomic DNA was subjected to random partialdigestion with DNase I, EcoRI linkers were added to the digestionproducts, and DNA fragments of 4-8 kb were isolated by agarose gelelectrophoresis and electroelution. The DNA fragments were then ethanolprecipitated and ligated into EcoRI-digested λgt11 arms. The ligationmixture was packaged into λ heads and the packaging mixture was used toinfect E. coli. Approximately 5×10⁶ recombinants were obtained.

EXAMPLE 1.

Isolation of BCG OMP DCase gene by complementation and plasmid DNAmanipulation. The BCG recombinant library was used to transform the E.coli strain Y1107. Twenty-one transformants capable of growing in theabsence of uracil were isolated, of which six were chosen for furtherevaluation by restriction analysis. Plasmid DNA was isolated by alkalinelysis from cells grown in liquid culture, and restriction analysisindicated that all of these plasmids contained the same or very similarinsert DNAs. One of these clones (pY6006) was used for further study(see FIG. 1). A 0.6 kb BamHI DNA fragment from pY6006 was used to screenthe λgt11 library, leading to the isolation of phage Y3030. This phagecarries a 5.6 kb EcoRI BCG DNA insert containing the OMP DCase gene.This insert DNA was subcloned into pGEMz(f+) to generate pY6011. The 4.4kb SacI-EcoRI fragment of the Y3030 insert was subcloned into pUC19 togenerate pY6014. Plasmid pY6015 was derived from pY6014 by replacinguraA sequences with the aph gene; a 1.15 kb HincII DNA fragmentcontaining uraA sequences was removed by partial HincII digestion ofpY6014 DNA, and it was replaced with a 1.3 kb BamHI fragment containingaph from pY6002 that was blunt-ended with Klenow.

DNA Seauence analysis. The M. Bovis BCG uraA gene was sequenced from the4.4 kb SacI-EcoRI fragment of the λgt11 phage Y3030 cloned into M13 inboth orientations. The same DNA fragment was subcloned into pUC19 togenerate pY6014 for further manipulation. Single strand DNA for sequenceanalysis was prepared from M13 grown in JM101 (Viera and Messing,Methods Enzymol., 153:3-11 1987). Both DNA strands were sequenced usingthe dideoxy-method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA74:5463-5467, 1977). Mycobacterial DNA has a high GC content, and twodifferent strategies were used to reduce band compression and otherartifacts due to high G+C content. A subset of the reactions was carriedout using Taq polymerase at high temperature (70° C.). In addition, dGTPand dITP were used in independent sequence reactions (Kimsey and Kaiser,J. Biol. Chem. 267:819-824, 1992).

RESULTS

Isolation of the BCG OMP decarboxylase gene by genetic complementation.The complementation strategy employed to isolate the BCG OMP DCase genewas similar to that employed previously to isolate the homologous genein M. smegmatis (Husson et al., J. Bacteriol. 172:519-524, 1990). Arecombinant library was constructed in the E. coli vector pUC19 usingsize selected BCG genomic DNA fragments from a partial SauIIIA digest.An E. coli pyrF mutant strain (Y1107) was transformed with this libraryand cells were plated on medium lacking uracil to select for uracilprototrophs, and on rich medium containing ampicillin to ascertain thetransformation frequency and to estimate the fraction of transformantsthat were able to complement the E. coli pyrF defect. Approximately0.05% of the cells transformed with the recombinant library becameuracil prototrophs. DNA clones were obtained from six colonies able togrow in the absence of uracil, and restriction analysis revealed thatthese clones contained the same insert DNA. One of these clones, pY6006,was subjected to further study (FIG. 1).

To identify, the portion of the 3.5 kb insert DNA pY6006 that wasresponsible for complementation, the 1.3-kb BamHI fragment of Tn903,which encodes amino-glycoside transferase (aph), was inserted intoseveral different sites in pY6006 insert DNA, the resultant plasmidswere reintroduced into the E. coli pyrF mutant strain, and the abilityof the new plasmids to complement the mutant phenotype was assessed asbefore (FIG. 1). One of the three plasmids with insertion mutations,pY6006B, lost the ability to complement the pyrF mutant phenotype,suggesting that sequences necessary for the complementing activity arelocated in the vicinity of the BamHI site that is disrupted in pY6006B.

Analysis of DNA sequences for the left end of pY6006 insert DNA (asdiagrammed in FIG. 1) revealed that the open reading frame of the pUC19lacZ gene in this plasmid continues uninterrupted into an open readingframe for a polypeptide similar in sequence to OMP decarboxylaseproteins. This preliminary data suggested that the left end of pY6006insert DNA encoded the amino-terminus of the BCG OMP decarboxylaseprotein.

For later experiments, it was important to have both the OMPdecarboxylase gene and a substantial amount of flanking sequences. Toobtain genomic DNA that contains both the OMP decarboxylase gene and itsflanking sequences, the 0.6 kb BamHI DNA fragment from pY6006 was usedto probe a λgt11 library, of M. bovis BCG DNA, as the λgt11 librarycontains insert DNA fragments whose size, on average, is larger (4-8 kb)than the plasmid library used to obtain pY6006. A lambda clone (Y3030)was isolated which contains a 5.6 kb EcoRI DNA insert that overlaps thatof pY6006. The 5.6 kb EcoRI DNA fragment, and a 4.4 kb SacI-EcoRIsubfragment, were subcloned into plasmid vectors to generate pY6011 andpY6014, respectively (FIG. 1). Both pY6011 and pY6014 were able tocomplement the defect of the E. coli pyrF mutant strain Y1107.

Sequence of the BCG OMP decarboxylase gene and flanking DNA. DNAfragments, from phage Y3030 insert DNA were subcloned into M13 vectorsand subjected to sequence analysis. Sequences were determined for bothDNA strands, and most of the sequence reactions were duplicated with ITPreplacing GTP to minimize artifacts due to the GC-rich nature ofmycobacterial DNA. FIGS. 2A-2D shows the sequences obtained for the BCGOMP decarboxylase gene (uraA) and for flanking DNA. The predicted BCGOMP decarboxylase protein sequence is 274 amino acids long, similar insize to other OMP decarboxylase proteins. When the BCG decarboxylaseprotein sequence was used to screen the available databases for similarsequences, the results revealed that the BCG protein is closely relatedto the Myxococcus xanthus OMP DCase (Kirnsey and Kaiser, J. Biol. Chem.267:819-824, 1992) and more distantly related to the other knownprokaryotic and eukaryotic OMP DCases. Comparison of the BCG and M.xanthus OMP decarboxylases reveals that 40% of the amino acid residuesare identical. In contrast, only 17% of the residues of the BCG and E.coli proteins and 22% of the amino acids of the M. xanthus and E. coliproteins are identical, although there are a substantial number ofconservative amino acid substitutions among these proteins. Therelationship of M. xanthus OMP decarboxylase to homologues in otherprokaryotes and in eukaryotes was recently described in some detail(Kimsey and Kaiser, J. Biol. Chem. 267:819-824, 1992). This comparativesequence analysis revealed that there are four regions which are morehighly conserved, and the predicted BCG OMP decarboxylase also sharesthis feature with the other homologues. It is interesting to note thatMycobacteria and Myxococci both have GC-rich genomes, but this alonedoes not account for the degree of sequence conservation between the OMPdecarboxylases from these two proaryotes; rather, the two genuses appearto be more closely related to one another than either is to the otherprokaryotes for which OMP decarboxylase sequence are available.

Further analysis of the BCG genomic DNA sequences revealed that the 1.7kb sequence upstream of OMP decarboxylase coding sequences contains asingle large open reading frame. This open reading frame has no apparentbeginning in the cloned DNA fragment, suggesting that it is the codingsequence for the carboxy-terminus of a larger protein. A screen of thesequence database revealed that the 497 amino acid residues of thepredicted protein are highly homologous to the carboxyl termini of thelarge subunit of carbamoyl phosphate synthase. For example, the 497amino acid carboxy terminus of the putative M. bovis BCG protein was 46%identical to the comparable segment of the E. coli carbarnoyl phosphatesynthase subunit, which is encoded by the carB gene (Nyunoya and Lusty,Proc. Natl. Acad. Sci. USA 80:4629-4633, 1983). Thus, the BCG carB geneappears to be located just upstream of uraA. This is interesting becauseboth carbamoyl phosphate synthase and OMP decarboxylase are involved inpyrimidine biosynthesis. Carbamoyl phosphate synthase catalyzes thefirst reaction in pyrimidine biosynthesis, the production of carbamoylphosphate, while OMP decarboxylase catalyzes the last step in thebiosynthesis of UMP.

Analysis of BCG DNA sequences downstream of the uraA gene revealed asingle large open reading frame that continues through the right end ofthe sequenced DNA fragment. This open reading frame predicts a proteinof 501 amino acids. A search of the computer database revealed that theprotein predicted by this ORF is similar to previously describedproteins from M. tuberculosis and M. leprae. The predicted BCG proteinis similar to a putative M. tuberculosis antigen encoded downstream ofthe gene for the 65 kDa antigen (Shinnick, T. M., J. Bacteriol.169:1080-1088, 1987) and to a M. leprae antigen that may be an integralmembrane protein (Vega-Lopez et al., Infect. Immun. 61:2145-2154, 1993).

Southern analysis with whole genomic DNA revealed that there is a singlecopy of the uraA gene and flanking DNA in the BCG genome (see below).The relative positions of the BCG uraA gene and the portions of othergenes identified through sequence analysis are summarized graphically inFIG. 1. The position of OMP decarboxylase sequences is consistent withthe genetic analysis described above. The aph insertion mutations inplasmid pY6006 that adversely affected complementation of the E. coliOMP decarboxylase mutant occurred within OMP decarboxylase codingsequences. Conversely, the aph insertion mutations that did not affectcomplementation of the E. coli OMP decarboxylase mutant occurred outsideof the BCG OMP decarboxylase coding sequences.

EXAMPLE 2

BCG transformation. BCG Pasteur (ATCC) was grown in log phase to anOD₆₀₀ of 0.5 in Middlebrook medium. BCG cells were harvested bycentrifugation and washed twice with PBS (phosphate bufered saline) andresuspended in 1 mM MgCl (pH 7.2), 10% sucrose, 15% glycerol at aconcentration of 10 OD₆₀₀ per ml. 0.4 ml of BCG cells was mixed with 2ug of plasmid DNA and electroporated in a 0.2 cm cuvette.Electroporation settings were 2.5 kV potential and 25 μF capacitance.After electroporation, cells were resuspended in 10 ml Middlebrookmedium and incubated at 37° C. for 2 hours before plating on Middlebrookagar containing 20 ug/ml kanamycin and, in some experiments, withuracil.

Southern blot analysis. Genomic DNAs from BCG strains were isolated asdescribed above, digested with restiction enzymes, subjected to agarosegel electrophoresis in the presense of ethidium bromide, transfered tonitrocellulose, and probed with DNA labelled with 32P by random priming,all by standard procedure (Ausubel et al., Current protocol in molecularbiology (1987). Green Publishing Associates and Wiley Interscience).

Introduction of foreign DNA into the BCG genome. Previous attempts toobtain homologous recombination in M. bovis BCG have apparently not beensuccessful (Kalpana et al., Proc. Natl. Acad. Sci. USA 88:5433-5447,1991; Young and Cole, J. Bacteriol. 175:1-6, 1993). It is possible thatthe efficiency of transformation has an influence on the ability toobtain homologous recombination. To maximize the transformationefficiency of BCG, we investigated the effect of adding glycine to theculture medium prior to harvesting cells for electroporation, as thepresence of 1.5% glycine can affect the integrity of the cell wall andit seems to improve transformation efficiency in M. smegmatis (Mizuguchiand Takunaga, "Spheroplasts of Mycobacteria. 2. Infection of Phage andIts DNA on Glycine Treated Mycobacteria and Spheroplasts", Med. Biol.,77:57 1968). In addition, we compared the efficiency of electroporationof BCG cells in water relative to buffer. The autonomously replicatingplasmid pYUB12 (snapper et al., Mol. Microbiol. 4:1911-1919, 1988) wasused to determine how these variables affected the relative efficienciesof transformation. The results are summarized in the Table underExperiment 1. Transformation efficiencies were improved substantially byexposing cultures to 1.5% glycine for 24 hours prior to harvest, and byperforming the electroporation in water rather than in buffer.

                  TABLE                                                           ______________________________________                                        BCG Transformation Efficiencies                                                                  Electro-                                                   Trans-   Glycine   poration  Transformants/ug DNA                             forming DNA.sup.a                                                                      Treatment.sup.b                                                                         Medium.sup.c                                                                            Expt 1                                                                              Expt 2                                                                              Expt 3                               ______________________________________                                        pYUB12   -         Buffer     50   --    --                                   pYUB12   +         Buffer    250   --    --                                   pYUB12   -         Water     500   --    --                                   pYUB12   +         Water      10.sup.4                                                                           10.sup.4                                                                             10.sup.5                            None     +         Water      8     6     35                                  p6015 (I)                                                                              --        Buffer    --     4    --                                   p6015 (I)                                                                              +         Buffer    --    22    --                                   p6015 (I)                                                                              -         Water     --    39    --                                   p6015 (I)                                                                              +         Water     --    98    500                                  ______________________________________                                         .sup.a The intact autonomously replicating plasmid pYUB12 was used as a       control and the linear insert DNA of plasmid pY6015  pY6015 (I)! was used     as integrating DNA.                                                           .sup.b Glycine was added to 1.5% to BCG cultures 24 hours prior to            transformation.                                                               .sup.c The buffer is 1 mM MgCl (pH 7.2), 10% sucrose, 15% glycerol.      

Experiments with linearized DNA molecules in yeast indicate that theends of linear DNA molecules are recombinogenic; these ends mayfacilitate homologous integration by invading genomic DNA at homologoussites to initiate recombination (Rothstein, R., Meth., Enzymol.194:281-301 (1988)). The sequenced 4.4 kb BCG DNA fragment containingUraA was used to investigate whether cloned DNA sequences couldintegrate at the homologous locus in M. bovis BCG. To mark the DNAfragment, the OMP decarboxylase coding sequence was replaced with akanomycin-resistance gene (aph) to create pY6015 (FIG. 3). This leftintact approximately 1.5 kb of UraA flanking sequences that could beused to direct homologous integration. The transformation experimentdescribed above for plasmid pYUB12 was repeated with pY6015 insert DNA,and the results are summarized in the Table under Experiment 2. Again,transformation efficiencies were improved substantially by exposingcultures to 1.5% glycine for 24 hours prior to harvest, and byperforming the electroporation in water rather than in buffer. However,because the transformation efficiencies obtained with the linear DNAwere low, we made one additional attempt to improve these efficiencies.

Cultures of M. bovis BCG and other slow growing mycobacteria containlarge numbers of cells that are inviable or that have an exceedinglylong lag time after plating. Some investigators have suggested thatmycobacterial cells have an unusual ability to enter and maintain adormant state, even when nutrients are available (Young and Cole,"Leprosy, Tuberculosis, and the New Genetics", J. Bacteriol., 175:1-61993). We reasoned that maintenance of BCG cultures in mid-log growthmight maximize the fraction of cells that were undergoing DNA synthesisand were competent to take up DNA and to incorporate it into homologoussites in the genome. A third experiment was performed, in which BCGcultures were diluted approximately 1:4 every two days over a two-monthperiod to ensure persistent log-phase growth before transformation. Theresults in the Table indicate that this approach produces a significantincrease in the number of transformants obtained with either theautonomously replicating vector or the linear DNA fragment.

Ten of the BCG colonies obtained in the third experiment were selectedfor further study after growing to adequate size for picking (24 daysafter plating). The ten transformants were colony purified, and DNA wasprepared from each. DNA preparations from the wild type strain and theten transformants were digested with a variety of restrictionendonucleases and Southern analysis revealed that thekanomycin-resistant BCG transformants all contained vector DNAintegrated into the genome. In two of the ten transformants, thetransforming DNA had integrated at the homologous locus. FIG. 5 showsrepresentative results from Southern analysis of the wild type strainand one of the BCG recombinants in which the cloned DNA integrated atthe homologous locus.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4394 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGCTCGACCCCGCCGCCGAAACAGAGGTGGCCCCGCAGACCGAAAGGCCCAAGGTGCTG60                ATCCTCGGTTCGGGGCCCAATCGGATCGGCCAGGGTATCGAGTTCGACTACAGCTGCGTA120               CACGCGGCAACCACGTTGAGCCAGGCTGGCTTTGAGACCGTGATGGTCAACTGCAACCCG180               GAGACCATGGTGTCCACCGACTTCGACACCGCGGACAGGTTGTACTTCGAGCCGTTGACG240               TTCGAGGACGTCTTGGAGGTCTACCACGCCGAAATGGAATCCGGTAGCGGTGGCCCGGGA300               GTGGCCGGCGTCATCGTGCAGCTCGGCGGCCAGACCCCGCTCGGCTGGCGCACCGGCTCG360               CCGACGCCGGGTCCCGCTCGTGGGCACCCACCGGAGGCCATCGACCTGGCCGAGGATGCG420               GCCGTTCGGCGACCTGCTGAGCGAGGACTGCCGGCGCCAAAGTACGGCACCGCAACCACT480               TTCGCCCAGGCCCGCCGGATCGCCGAGGAGATCGGCTATCCGGTGCTGGTGCGGCCGTCG540               TATGTGCTCGGTGGTCGCGGCATGGAGATCGTGTATGACGAAGAAACGTTGCAGGGCTAC600               ATCACCCGCGCCACTCAGCTATCCCCCGAACACCCGGTGCTCGTGCACCGCTTCCTCGAG660               GACGCGGTCGAGATCGACGTCGACGCTCTGTGTGATGGCGCCGAGGTCTATATCGGCGGA720               ATCATGGAGCACATCGAGGAGGCCGGCATCCACTCCGGTGACTCGGCCTGTGCGCTGCCA780               CCGGTCACGTTGGGCCGCAGCGACATCGAGAAGGTGCGTAAGGCCACTGAAGCCATTGCG840               CATGGCATCGGCGTGGTGGGGCTGCTCAACGTGCAGTCCGCGCTCAAGGATGACGTGCTC900               TACGTCCTGGAAGCCAACCCGAGAGCGAGCCGTACCGTTCCGTTTGTATCCAAGGCCACA960               GCGGTGCCACTCGCCAAGGCATGCGCCCGGATCATGTTGGGCGCCACCATTGCCCAGCTG1020              CGCGCCGAAGGCTTGCTGGCGGTCACCGGGGATGGCGCCCACGCGGCGCGAAACGCCCCC1080              ATCGCGGTCAACCAGGCCGTGTTGCCGTTTCACCGGTTCCGGCGCGCCGACGGGGCCGCC1140              ATCGACTCGCTACTCGGCCCGGAGATGAAATCGACCGGCGAGGTGATGGGCATCGACCGC1200              GACTTCGGCAGCCGGTTCGCCAAGAGCCAGACCGCCGCCTACGGGTCGCTGCCGGCCCAG1260              GGCACAGTGTTCGTGTCGGTGGCCAACCGGGACAAGCGGTCGCTGGTGTTTCCGGTCAAA1320              CCGATTGGCCCACCTGGGTTTTCGCGTCCTTGCCACCGAAGCACCGCAGAGATCTTGCGC1380              CGCAACGGTATTCCCTGCGACGACGTCCGCAAACATTTCGAGCCGGCGCAGCCCGGCCGC1440              CCCACAATGTCGGCGGTGGACGCGATCCGAGCCGGCGAGGTCAACATGGTGATCAACACT1500              CCCTATGGCAACTCCGGTCCGCGCATCGACGGCTATGAGATCCGTTCGGCGGCGGTGGCC1560              GGCAACATCCCGTGCATCACCACGGTGCAGGGCGCATCCGCCGCCGTGCAGGGGATAGAG1620              GCCGGGATCCGCGGCGACATCGGGGTGCGCTCCCTGCAGGAGCTGCACCGGGTGATCGGG1680              GGCGTCGAGCGGTGACCGGGTTCGGTCTCCGGTTGGCCGAGGCAAAGGCACGCCGCGGCC1740              CGTTGTGTCTGGGCATCGATCCGCATCCCGAGCTGCTGCGGGGCTGGGATCTGGCGACCA1800              CGGCCGACGGGCTGGCCGCGTTCTGCGACATCTGCGTACGGGCCTTCGCTGATTTCGCGG1860              TGGTCAAACCGCAGGTGGCGTTTTTTGAGTCATACGGGGCTGCCGGATTCGCGGTGCTGG1920              AGCGCACCATCGCGGAACTGCGGGCCGCAGACGTGCTGGTGTTGGCCGACGCCAAGCGCG1980              GCGACATTGGGGCGACCATGTCGGCGTATGCGACGGCCTGGGTGGGCGACTCGCCGCTGG2040              CCGCCGACGCCGTGACGGCCTCGCCCTATTTGGGCTTCGGTTCGCTGCGGCCGCTGCTAG2100              AGGTCGCGGCCGCCCACGGCCGAGGGGTGTTCGTGCTGGCGGCCACCTCCAATCCCGAGG2160              GTGCGGCGGTGCAGAATGCCGCCGCCGACGGCCGCAGCGTGGCCCAGTTGGTCGTGGACC2220              AGGTGGGGGCGGCCAACGAGGCGGCAGGACCCGGGCCCGGATCCATCGGCGTGGTCGTCG2280              GCGCAACGGCGCCACAGGCCCCCGATCTCAGCGCCTTCACCGGGCCGGTGCTGGTGCCCG2340              GCGTGGGGGTGCAGGGCGGGCGCCCGGAGGCGCTGGGCGGTCTGGGCGGGGCCGCATCGA2400              GCCAGCTGTTGCCCGCGGTGGCGCGCGAGGTCTTGCGGGCCGGCCCCGGCGTGCCCGAAT2460              TGCGCGCCGCGGGCGAACGGATGCGCGATGCCGTCGCCTATCTCGCTGCCGTGTAGCGGG2520              TGCCCTGCCACCGCGCCGCTAAATCCCACCAGCATGGGGTGGTGAGCCCAGCGCTCGTGT2580              GACCAAACTCACCGCCCTGGGCCGTCGTCACGCTGTGTTAACCTCTCGTTCAAATGATAT2640              TCATATTCAATAGTGGCGCTAAGTGTCCGGTTGAATCCCCGTTGAACCCCCAACAGATGG2700              AGTCTGTGTCGTGACGTTGCGAGTCGTTCCCGAAAGCCTGGCAGGCGCCAGCGCTGCCAT2760              CGAAGCAGTGACCGCTCGCCTGGCCGCCGCGCACGCCGCGGCGGCCCCGTTTATCGCGGC2820              GGTCATCCCGCCTGGGTCCGACTCGGTTTCGGTGTGCAACGCCGTTGAGTTCAGCGTTCA2880              CGGTAGTCAGCATGTGGCAATGGCCGCTCAGGGGGTTGAGGAGCTCGGCCGCTCGGGGGT2940              CGGGGTGGCCGAATCGGGTGCCAGTTATGCCGCTAGGATGCGCTGGCGGCGGCGTCGTAT3000              CTCAGCGGTGGGCTATGACCGAGCCGTGGATAGCCTTCCCTCCCGAGGTGCACTCGGCGA3060              TGCTGAACTACGGTGCGGGCGTTGGGCCGATGTTGATCTCCGCCACGCAGAATGGGGAGC3120              TCAGCGCCCAATACGCAGAAGCGGCATCCGAGGTCGAGGAATTGTTGGGGGTGGTGGCCT3180              CCGAGGGATGGCAGGGGCAAGCCGCCGAGGCGTTAGTCGCCGCGTACATGCCGTTTCTGG3240              CGTGGCTGATCCAAGCCAGCGCCGACTGCGTGGAAATGGCCGCCCAGCAACACGCCGTCA3300              TCGAGGCCTACACTGCCGCGGTAGAGCTGATGCCTACTCAGGTCGAACTGGCCGCCAACC3360              AAATCAAGCTCGCGGTGTTGGTAGCGACCAATTTCTTTGGCATCAACACCATTCCCATTG3420              CGATCAATGAGGCCGAGTACGTGGAGATGTGGGTTCGGGCCGCCACCACGATGGCGACCT3480              ATTCAACAGTCTCCAGATCGGCGCTCTCCGCGATGCCGCACACCAGCCCCCCGCCGCTGA3540              TCCTGAAATCCGATGAACTGCTCCCCGACACCGGGGAGGACTCCGATGAAGACGGCCACA3600              ACCATGGCGGTCACAGTCATGGCGGTCACGCCAGGATGATCGATAACTTCTTTGCCGAAA3660              TCCTGCGTGGCGTCAGCGCGGGCCGCATTGTTTGGGACCCCGTCAACGGCACCCTCAACG3720              GACTCGACTACGACGATTACGTCTACCCCGGTCACGCGATCTGGTGGCTGGCTCGAGGCC3780              TCGAGTTTTTTCAGGATGGTGAACAATTTGGCGAACTGTTGTTCACCAATCCGACTGGGG3840              CTTTTCAGTTCCTCCTCTACGTCGTTGTGGTGGATTTGCCGACGCACATAGCCCAGATCG3900              CTACCTGGCTGGGCCAGTACCCGCAGTTGCTGTCGGCTGCCCTCACTGGCGTCATCGCCC3960              ACCTGGGAGCAATAACTGGTTTGGCGGGCCTATCCGGCCTGAGCGCCATTCCGTCTGCTG4020              CGATACCCGCCGTTGTACCGGAGCTGACACCCGTCGCGGCCGCGCCGCCTATGTTGGCGG4080              TCGCCGGGGTGGGCCCTGCAGTCGCCGCGCCGGGCATGCTCCCCGCCTCAGCACCCGCAC4140              CGGCGGCAGCGGCCGGCGCCACCGCAGCCGGCCCGACGCCGCCGGCGACTGGTTTCGGAG4200              GGCTTCCCGCCCTACCTGGTCGGCGGTGGCGGCCCAGGAATAGGGTTCGGCTCGGGACAG4260              TCGGCCCACGCCAAGGCCGCGGCGTCCGATTCCGCTGCAGCCGAGTCGGCGGCCCAGGCC4320              TCGGCGCGTGCGCAGGCGCGTGCTGCACGGCGGGGCCGCTCGGCGGCAAGGCACGTGGCC4380              ATCGTGACGAATTC4394                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1271 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluLeuAspProAlaAlaGluThrGluValAlaProGlnThrGluArg                              151015                                                                        ProLysValLeuIleLeuGlySerGlyProAsnArgIleGlyGlnGly                              202530                                                                        IleGluPheAspTyrSerCysValHisAlaAlaThrThrLeuSerGln                              354045                                                                        AlaGlyPheGluThrValMetValAsnCysAsnProGluThrMetVal                              505560                                                                        SerThrAspPheAspThrAlaAspArgLeuTyrPheGluProLeuThr                              65707580                                                                      PheGluAspValLeuGluValTyrHisAlaGluMetGluSerGlySer                              859095                                                                        GlyGlyProGlyValAlaGlyValIleValGlnLeuGlyGlyGlnThr                              100105110                                                                     ProLeuGlyTrpArgThrGlySerProThrProGlyProAlaArgGly                              115120125                                                                     HisProProGluAlaIleAspLeuAlaGluAspAlaAlaValArgArg                              130135140                                                                     ProAlaGluArgGlyLeuProAlaProLysTyrGlyThrAlaThrThr                              145150155160                                                                  PheAlaGlnAlaArgArgIleAlaGluGluIleGlyTyrProValLeu                              165170175                                                                     ValArgProSerTyrValLeuGlyGlyArgGlyMetGluIleValTyr                              180185190                                                                     AspGluGluThrLeuGlnGlyTyrIleThrArgAlaThrGlnLeuSer                              195200205                                                                     ProGluHisProValLeuValHisArgPheLeuGluAspAlaValGlu                              210215220                                                                     IleAspValAspAlaLeuCysAspGlyAlaGluValTyrIleGlyGly                              225230235240                                                                  IleMetGluHisIleGluGluAlaGlyIleHisSerGlyAspSerAla                              245250255                                                                     CysAlaLeuProProValThrLeuGlyArgSerAspIleGluLysVal                              260265270                                                                     ArgLysAlaThrGluAlaIleAlaHisGlyIleGlyValValGlyLeu                              275280285                                                                     LeuAsnValGlnSerAlaLeuLysAspAspValLeuTyrValLeuGlu                              290295300                                                                     AlaAsnProArgAlaSerArgThrValProPheValSerLysAlaThr                              305310315320                                                                  AlaValProLeuAlaLysAlaCysAlaArgIleMetLeuGlyAlaThr                              325330335                                                                     IleAlaGlnLeuArgAlaGluGlyLeuLeuAlaValThrGlyAspGly                              340345350                                                                     AlaHisAlaAlaArgAsnAlaProIleAlaValAsnGlnAlaValLeu                              355360365                                                                     ProPheHisArgPheArgArgAlaAspGlyAlaAlaIleAspSerLeu                              370375380                                                                     LeuGlyProGluMetLysSerThrGlyGluValMetGlyIleAspArg                              385390395400                                                                  AspPheGlySerArgPheAlaLysSerGlnThrAlaAlaTyrGlySer                              405410415                                                                     LeuProAlaGlnGlyThrValPheValSerValAlaAsnArgAspLys                              420425430                                                                     ArgSerLeuValPheProValLysArgLeuAlaHisLeuGlyPheArg                              435440445                                                                     ValLeuAlaThrGluAlaProGlnArgSerCysAlaAlaThrValPhe                              450455460                                                                     ProAlaThrThrSerAlaAsnIleSerSerArgArgSerProAlaAla                              465470475480                                                                  ProGlnCysArgArgTrpThrArgSerGluProAlaArgSerThrTrp                              485490495                                                                     MetThrGlyPheGlyLeuArgLeuAlaGluAlaLysAlaArgArgGly                              500505510                                                                     ProLeuCysLeuGlyIleAspProHisProGluLeuLeuArgGlyTrp                              515520525                                                                     AspLeuAlaThrThrAlaAspGlyLeuAlaAlaPheCysAspIleCys                              530535540                                                                     ValArgAlaPheAlaAspPheAlaValValLysProGlnValAlaPhe                              545550555560                                                                  PheGluSerTyrGlyAlaAlaGlyPheAlaValLeuGluArgThrIle                              565570575                                                                     AlaGluLeuArgAlaAlaAspValLeuValLeuAlaAspAlaLysArg                              580585590                                                                     GlyAspIleGlyAlaThrMetSerAlaTyrAlaThrAlaTrpValGly                              595600605                                                                     AspSerProLeuAlaAlaAspAlaValThrAlaSerProTyrLeuGly                              610615620                                                                     PheGlySerLeuArgProLeuLeuGluValAlaAlaAlaHisGlyArg                              625630635640                                                                  GlyValPheValLeuAlaAlaThrSerAsnProGluGlyAlaAlaVal                              645650655                                                                     GlnAsnAlaAlaAlaAspGlyArgSerValAlaGlnLeuValValAsp                              660665670                                                                     GlnValGlyAlaAlaAsnGluAlaAlaGlyProGlyProGlySerIle                              675680685                                                                     GlyValValValGlyAlaThrAlaProGlnAlaProAspLeuSerAla                              690695700                                                                     PheThrGlyProValLeuValProGlyValGlyValGlnGlyGlyArg                              705710715720                                                                  ProGluAlaLeuGlyGlyLeuGlyGlyAlaAlaSerSerGlnLeuLeu                              725730735                                                                     ProAlaValAlaArgGluValLeuArgAlaGlyProGlyValProGlu                              740745750                                                                     LeuArgAlaAlaGlyGluArgMetArgAspAlaValAlaTyrLeuAla                              755760765                                                                     AlaValMetTrpGlnTrpProLeuArgGlyLeuArgSerSerAlaAla                              770775780                                                                     ArgGlySerGlyTrpProAsnArgValProValMetProLeuGlyCys                              785790795800                                                                  AlaGlyGlyGlyValValSerGlnArgTrpAlaMetThrGluProTrp                              805810815                                                                     IleAlaPheProProGluValHisSerAlaMetLeuAsnTyrGlyAla                              820825830                                                                     GlyValGlyProMetLeuIleSerAlaThrGlnAsnGlyGluLeuSer                              835840845                                                                     AlaGlnTyrAlaGluAlaAlaSerGluValGluGluLeuLeuGlyVal                              850855860                                                                     ValAlaSerGluGlyTrpGlnGlyGlnAlaAlaGluAlaLeuValAla                              865870875880                                                                  AlaTyrMetProPheLeuAlaTrpLeuIleGlnAlaSerAlaAspCys                              885890895                                                                     ValGluMetAlaAlaGlnGlnHisAlaValIleGluAlaTyrThrAla                              900905910                                                                     AlaValGluLeuMetProThrGlnValGluLeuAlaAlaAsnGlnIle                              915920925                                                                     LysLeuAlaValLeuValAlaThrAsnPhePheGlyIleAsnThrIle                              930935940                                                                     ProIleAlaIleAsnGluAlaGluTyrValGluMetTrpValArgAla                              945950955960                                                                  AlaThrThrMetAlaThrTyrSerThrValSerArgSerAlaLeuSer                              965970975                                                                     AlaMetProHisThrSerProProProLeuIleLeuLysSerAspGlu                              980985990                                                                     LeuLeuProAspThrGlyGluAspSerAspGluAspGlyHisAsnHis                              99510001005                                                                   GlyGlyHisSerHisGlyGlyHisAlaArgMetIleAspAsnPhePhe                              101010151020                                                                  AlaGluIleLeuArgGlyValSerAlaGlyArgIleValTrpAspPro                              1025103010351040                                                              ValAsnGlyThrLeuAsnGlyLeuAspTyrAspAspTyrValTyrPro                              104510501055                                                                  GlyHisAlaIleTrpTrpLeuAlaArgGlyLeuGluPhePheGlnAsp                              106010651070                                                                  GlyGluGlnPheGlyGluLeuLeuPheThrAsnProThrGlyAlaPhe                              107510801085                                                                  GlnPheLeuLeuTyrValValValValAspLeuProThrHisIleAla                              109010951100                                                                  GlnIleAlaThrTrpLeuGlyGlnTyrProGlnLeuLeuSerAlaAla                              1105111011151120                                                              LeuThrGlyValIleAlaHisLeuGlyAlaIleThrGlyLeuAlaGly                              112511301135                                                                  LeuSerGlyLeuSerAlaIleProSerAlaAlaIleProAlaValVal                              114011451150                                                                  ProGluLeuThrProValAlaAlaAlaProProMetLeuAlaValAla                              115511601165                                                                  GlyValGlyProAlaValAlaAlaProGlyMetLeuProAlaSerAla                              117011751180                                                                  ProAlaProAlaAlaAlaAlaGlyAlaThrAlaAlaGlyProThrPro                              1185119011951200                                                              ProAlaThrGlyPheGlyGlyLeuProAlaLeuProGlyArgArgTrp                              120512101215                                                                  ArgProArgAsnArgValArgLeuGlyThrValGlyProArgGlnGly                              122012251230                                                                  ArgGlyValArgPheArgCysSerArgValGlyGlyProGlyLeuGly                              123512401245                                                                  AlaCysAlaGlyAlaCysCysThrAlaGlyProLeuGlyGlyLysAla                              125012551260                                                                  ArgGlyHisArgAspGluPhe                                                         12651270                                                                      __________________________________________________________________________

We claim:
 1. A method of producing a homologously recombinantslow-growing mycobacterium having heterologous DNA which encodes aproduct to be expressed by the mycobacterium incorporated into genomicDNA thereof at a homologous locus, comprising the steps of:a) combininga slow-growing mycobacterium and heterologous DNA to be transformed intothe slow-growing mycobacterium, the heterologous DNA comprising DNAhomologous to genomic DNA of the slow-growing mycobacterium, therebyproducing a combination; and b) subjecting the combination produced instep (a) to electroporation in water, under conditions sufficient forintroduction of the heterologous DNA into the genomic DNA of theslow-growing mycobacterium through homologous recombination between theheterologous DNA and genomic DNA at a homologous locus,wherein ahomologously recombinant slow-growing mycobacterium having heterologousDNA incorporated into genomic DNA thereof at a homologous locus isproduced and wherein said mycobacterium expresses the product encoded bythe heterologous DNA.
 2. The method of claim 1 wherein the slow-growingmycobacterium of (a) has been exposed to glycine, prior to beingcombined with the heterologous DNA.
 3. The method of claim 2 wherein theslow-growing mycobacterium is exposed to approximately 1.5% glycinepresent in culture medium in which the slow-growing mycobacterium isgrowing.
 4. The method of claim 1 in which the slow-growingmycobacterium is continuously propagated in mid-log phase.
 5. The methodof claim 1 wherein the slow-growing mycobacterium is selected from thegroup consisting of: Mycobacterium bovis BCG, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium avium, Mycobacteriumafricanum and Mycobacterium intracellulare.
 6. The method of claim 1wherein the heterologous DNA additionally comprises DNA which is nothomologous to genomic DNA of the slow-growing mycobacterium combined instep (a) with the heterologous DNA.
 7. The method of claim 6 wherein theslow-growing mycobacterium is Mycobacterium bovis BCG and the DNAhomologous to genomic DNA of the slow-growing mycobacterium is DNAcontained in the Mycobacterium bovis BCG orotidine-5- monophosphatedecarboxylase gene locus or flanking sequences thereof.
 8. A method ofproducing a viable homologously recombinant slow-growing mycobacteriumhaving heterologous DNA incorporated into genomic DNA thereof at ahomologous locus, comprising the steps of:a) combining a slow-growingmycobacterium and heterologous DNA to be transformed into theslow-growing mycobacterium, the heterologous DNA comprising DNAhomologous to genomic DNA of the slow-growing mycobacterium, therebyproducing a combination; and b) subjecting the combination produced instep (a) to electroporation in water, under conditions sufficient forintroduction of the heterologous DNA into the genomic DNA of theslow-growing mycobacterium through homologous recombination between theheterologous DNA and genomic DNA at a homologous locus,wherein a viablehomologously recombinant slow-growing mycobacterium having heterologousDNA incorporated into genomic DNA thereof at a homologous locus isproduced.
 9. The method of claim 8 wherein the slow-growingmycobacterium of (a) has been exposed to glycine, prior to beingcombined with the heterologous DNA.
 10. The method of claim 9 whereinthe slow-growing mycobacterium is exposed to approximately 1.5% glycinepresent in culture medium in which the slow-growing mycobacterium isgrowing.
 11. The method of claim 9 wherein the slow-growingmycobacterium is continuously propagated in mid-log phase.
 12. Themethod of claim 9 wherein the slow-growing mycobacterium is selectedfrom the group consisting of:Mycobacterium bovis BCG, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium avium, Mycobacteriumafricanum and Mycobacterium intracellulare.
 13. The method of claim 9wherein the heterologous DNA additionally comprises DNA which is nothomologous to genomic DNA of the slow-growing mycobacterium combined instep (a) with the heterologous DNA.
 14. The method of claim 13 whereinthe slow-growing mycobacterium is Mycobacterium bovis BCG and the DNAhomologous to genomic DNA of the slow-growing mycobacterium is DNAcontained in the Mycobacterium bovis BCG orotidine-5-monophosphatedecarboxylase gene locus or flanking sequences thereof.
 15. A method oftransforming a slow-growing mycobacterium with heterologous DNA toproduce a recombinant slow-growing mycobacterium having heterologous DNAintegrated into genomic DNA thereof, comprising the steps of:a)combining a slow-growing mycobacterium and heterologous DNA to betransformed into the slow-growing mycobacterium, the heterologous DNAcomprising DNA homologous to genomic DNA of the slow-growingmycobacterium, thereby producing a combination; and b) subjecting thecombination produced in step (a) to electroporation, under conditionssufficient for introduction of the heterologous DNA into the genomic DNAof the slow-growing mycobacterium through homologous recombinationbetween the heterologous DNA and genomic DNA at a homologouslocus,wherein a recombinant slow-growing mycobacterium havingheterologous DNA integrated into genomic DNA is produced.
 16. The methodof claim 15 wherein the slow-growing mycobacterium of step (a) has beenexposed to glycine, prior to being combined with the heterologous DNA.17. The method of claim 16 wherein the slow-growing mycobacterium isexposed to approximately 1.5% glycine present in culture medium in whichthe slow-growing mycobacterium is growing.
 18. The method of claim 16wherein the slow-growing mycobacterium is continuously propagated inmid-log phase, prior to being combined with the heterologous DNA. 19.The method of claim 16 wherein the slow-growing mycobacterium isselected from the group consisting of:Mycobacterium bovis BCG,Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium avium,Mycobacterium africanum and Mycobacterium intracellulare.
 20. The methodof claim 19 wherein the heterologous DNA additionally comprises DNAwhich is not homologous to genomic DNA of the slow-growing mycobacteriumcombined in step (a) with the heterologous DNA.
 21. The method of claim20 wherein the slow-growing mycobacterium is Mycobacterium bovis BCG andthe DNA homologous to genomic DNA of the slow-growing mycobacterium isDNA contained in the Mycobacterium bovis BCG orotidine-5-monophosphatedecarboxylase gene locus or flanking sequences thereof.
 22. A method ofproducing a viable homologously recombinant slow-growing mycobacteriumhaving heterologous DNA incorporated into genomic DNA thereof at ahomologous locus, comprising the steps of:a) combining a slow-growingmycobacterium and heterologous DNA to be transformed into theslow-growing mycobacterium, the heterologous DNA comprising DNAhomologous to genomic DNA of the slow-growing mycobacterium, therebyproducing a combination; and b) subjecting the combination produced instep (a) to electroporation, under conditions sufficient forintroduction of the heterologous DNA into the genomic DNA of theslow-growing mycobacterium through homologous recombination between theheterologous DNA and genomic DNA at a homologous locus,wherein a viablehomologously recombinant slow-growing mycobacterium having heterologousDNA incorporated into genomic DNA thereof at a homologous locus isproduced.
 23. The method of claim 22 wherein the slow-growingmycobacterium of (a) has been exposed to glycine, prior to beingcombined with the heterologous DNA.
 24. The method of claim 23 whereinthe slow-growing mycobacterium is exposed to approximately 1.5% glycinepresent in culture medium in which the slow-growing mycobacterium isgrowing.
 25. The method of claim 23 wherein the slow-growingmycobacterium is continuously propagated in mid-log phase.
 26. Themethod of claim 23 wherein the slow-growing mycobacterium is selectedfrom the group consisting of:Mycobacterium bovis BCG, Mycobacteriumtuberculosis, Mycobacterium leprae, Mycobacterium avium, Mycobacteriumafricanum and Mycobacterium intracellulare.
 27. The method of claim 23wherein the heterologous DNA additionally comprises DNA which is nothomologous to genomic DNA of the slow-growing mycobacterium combined instep (a) with the heterologous DNA.
 28. The method of claim 27 whereinthe slow-growing mycobacterium is Mycobacterium bovis BCG and the DNAhomologous to genomic DNA of the slow-growing mycobacterium is DNAcontained in the Mycobacterium bovis BCG orotidine-5-monophosphatedecarboxylase gene locus or flanking sequences thereof.